Gene therapy for neuromuscular and neuromotor disorders

Abstract

The present invention provides methods for screening for nucleotide sequences encoding the capsid of adeno-associated virus ("AAV") particles capable of infecting neurons or subcompartments thereof in a subject, including neurons derived from induced pluripotent stem cells or embryonic stem cells. The present invention also provides AAV capsids, nucleotide sequences encoding the capsids, expression vectors, viral particles, cells, and kits for use in methods for treating neuromuscular or neuromotor disorders (e.g., spasticity). Sequences generated by these methods may lead to new gene therapies that offer high specificity that can be personalized to the subject because they are targeted to select neuronal populations, such as motor neurons that innervate muscle cells.

Description

[Technical Field]

[0001] The present invention relates, in general terms, to a method for screening adeno-associated virus ("AAV") capsids that target a defined population of neurons (e.g., motor neurons), and the method can be used to develop gene therapies for treating neuromuscular and neuromotor disorders (e.g., spasticity). [Background technology]

[0002] Symptoms of neurological disorders and diseases result from abnormal functioning of neurons and circuits in the brain and spinal cord. Current treatments aim to target this dysfunction and include relatively new areas of functional neurosurgery, such as deep brain stimulation, spinal cord stimulation, or the implantation of devices (e.g., pumps) for localized drug delivery. The key to the success of these treatments lies in the placement of the device implanted in the area of ​​neurological dysfunction. In other words, it is crucial to treat the dysfunctional neurons.

[0003] An alternative to device implantation is gene therapy, which involves inserting genes to modify neuronal function. However, both device implantation and genetic material insertion still face the problem of specificity. That is, many different types of neurons exist at the site where the device is implanted, and all of them could potentially be affected.

[0004] Another option is to use gene therapy that specifically targets neurons involved in circuit dysfunction. This can be done by combining three factors: (a) a promoter gene specific to the type of neuron, (b) inserting genetic material into a different location within the circuit, i.e., where the target neuron protrudes, so that the genetic material is sent back to the nerve cell body to modify its function, and (c) increasing the viral tropism towards a specific neuron type.

[0005] Recombinant adeno-associated vectors ("rAAVs") are important vectors for transferring genetic material into cells, and are the primary viral vectors used, particularly in gene therapy (Li and Sumulski, 2020). Several rAAV-based gene therapies are currently in use, such as Zolgensma for treating spinal muscular atrophy and Luxtern for treating subtype if retinal dystrophy, and many more are currently undergoing clinical trials.

[0006] AAV is a small virus belonging to the genus dependoparvovirus, containing single-stranded DNA up to approximately 4.9 kb. The AAV genome contains three capsid proteins, VP1, VP2, and VP3, all translated from a single mRNA molecule. In the wild type, multiple serotypes of AAV have been identified, each possessing a unique capsid gene sequence and therefore different tropisms; however, wild-type serotypes tend to infect multiple tissues and cell types. These serotypes are denoted by numbers, such as AAV1, AAV2, etc.

[0007] By modifying capsid sequences using DNA recombination, it is possible to generate non-natural sequences with properties and tropisms directed towards (or against) specific cells or tissues, and possessing properties and tropisms that allow them to evade the immune system (Vandenberghe et al., 2009). Generally, capsid modification can be achieved in two ways: random mutagenesis of existing capsid DNA sequences, or capsid shuffling (i.e., taking DNA sequences from multiple capsids and randomly shuffling parts of those sequences to create new capsids; Buning et al., 2015). These methods generate a large number of highly diverse capsids with potentially valuable properties. Once packaged into functional virions, they can be screened in animal tissues or cell cultures to select capsids targeted to specific tissues or cells. These capsid sequences can then be newly generated and combined with genes that have therapeutic potential in gene therapy.

[0008] This system, which screens capsid libraries to create novel gene therapy vectors, has been successfully applied in animals to derivatives of AAV9 that can cross the blood-brain barrier and infect neurons after systemic injection. rAAVs such as PhP.eB (described in Bedbrook et al., 2018) were created in this manner. In this system, an rAAV capsid library is injected into experimental animals, and then a target cell population (in this case, neurons) is collected and subjected to sequencing (by PCR or deep sequencing) to identify the AAV capsid sequences that infected those cells. These sequences can then be newly synthesized (de novo synthesis) and used in further experiments, or subjected to further mutagenesis and screening in a process known as directional evolution (to increase evolutionary pressure) (see Li and Sumulski, 2020). This type of approach has been used to create rAAVs targeted at dopaminergic neurons (Davidsson et al., 2019), striated muscle (Yang et al., 2009), and parts of the retina (Dalkara et al., 2013). rAAV capsids can also be evolved to reduce infection in vital organs such as the liver (Pulicherla et al., 2011). Finally, directional evolution of rAAV capsids can also be used to target subcompartments of individual cell types. For example, rAAV2-Retro was evolved to target synaptic terminals of mouse cortical neurons (Tervo et al., 2016).

[0009] In vivo studies have shown that directional evolution of AAV capsids can result in vectors with useful properties such as the ability to cross the blood-brain barrier and high neural tropism (Deverman et al., 2016 and EP3044318B1), or in particular, neural tropism towards dopaminergic neurons (Daviddson et al., 2019) or cardiomyocytes (Yang et al., 2009) (see Li and Sumulski, 2020 for review). Directional evolution involves the creation of a capsid library, which is a mixture of AAV vectors capsidized by random capsid sequences produced by error-prone PCR, capsid shuffling, or both. Subsequently, this library is applied to cell lines (e.g., Maheshri et al., 2006), undifferentiated stem cells (Asuri et al., 2012), or most commonly, experimental animals (e.g., Devermann et al., 2016; Li and Sumulski, 2020; patent US8632764B2; US20170166926A1; US9701984B2). Furthermore, there is a preliminary report from one group that produced cardiac-acting AAVs using iPSCs differentiated into cardiomyocytes.

[0010] While the construction of AAV capsid libraries can be achieved in numerous ways, screening these libraries relies primarily on the use of experimental animals. One major drawback of this method for producing rAAV, which are effective gene therapy vectors, is that the identified capsids are often species- or even strain-specific to the animal. Consequently, many of the improved features and functions of capsids developed in screening hosts (i.e., mice) do not readily transfer to non-human primates and humans (Hordeaux et al., 2018). This may be the main reason why engineered capsids have not yet widely replaced wild-type variants in clinical and preclinical studies (Davidsson et al., 2019).

[0011] One example of a neurological disorder or condition that can be treated as described above is spasticity. Spasticity is a neurological symptom that affects people with a variety of neurological disorders, including but not limited to multiple sclerosis, stroke, traumatic brain injury, spinal cord injury, and cerebral palsy. It is estimated that approximately 12 million people worldwide suffer from spasticity, with about 22% of patients unable to work because of the condition, almost all patients reporting an impact on their quality of life, and about 50% reporting a significant impact on their quality of life. Spasticity is caused by excessive excitation of muscles by motor neurons that have become "hyperexcitable" due to the disease.

[0012] Current treatments for spasticity include oral medications (e.g., baclofen), intramuscular injections of paralyzing agents (botulinum toxin), or surgical insertion of a pump that directly delivers the drug (e.g., baclofen) to the cerebrospinal fluid. Oral medications are often insufficient or cause significant adverse effects. Botox requires repeated administration several times a year throughout a person's life. Botox is often injected into only a limited number of muscles and can have long-term adverse effects. Baclofen pumps can be very effective in relieving symptoms, however, they are highly invasive as they require surgical intervention for implantation and replenishment 2-4 times a year, and must be replaced every 6-7 years. Furthermore, within the 6-7 year lifespan of the pump, 1 / 3-1 / 2 of patients may require additional surgery to correct problems. Each procedure also has a defined incidence of complications.

[0013] Therefore, there is an urgent need to provide novel gene therapy vectors that are specific to certain neurons and can be used in subjects such as human subjects to treat neuromuscular and neuromotor disorders such as spasticity. [Overview of the project]

[0014] The present invention includes gene therapy designed to alter the activity of targeted neurons. Success of this treatment occasionally requires efficient infection (or "transduction") of neurons after intramuscular injection. To the best of the inventors' knowledge, this has not been possible with previous treatments / inventions. In some preferred embodiments, the neurons are motor neurons. In other preferred embodiments, the neurons are sensory neurons, interneurons, or projection neurons.

[0015] To achieve this, the present invention includes a technological platform for identifying novel adeno-associated virus (AAV) capsids based on their ability to efficiently infect specific classes of neurons, such as motor neurons. This platform screens AAV capsid libraries using stem cells differentiated into these cell types and uses sequences obtained from these screenings to develop novel gene therapies targeted at selected populations of neurons or other human cells or tissues. Neurons can be defined anatomically, functionally, through gene expression, or by their involvement in a disease state (e.g., dopaminergic neurons in Parkinson's disease).

[0016] The present invention also includes a treatment method comprising injecting an AAV containing a novel AAV capsid into the affected muscle of a subject, after which these AAVs can infect the terminals of motor neurons, be transported to their cell bodies, and provide high specificity by specifically expressing the exogenous gene in the motor neurons innervating that muscle (see Figure 1).

[0017] Therefore, the present invention makes it possible to create a virus that accesses motor neurons after intramuscular injection and subsequently modifies gene expression in motor neurons, for the purpose of curing, alleviating symptoms, and / or improving the quality of life of patients with diseases affecting motor neurons.

[0018] The present invention also includes a treatment method comprising injecting an AAV containing a novel AAV capsid into the brain or spinal cord of a subject, after which these AAVs can infect the terminals of motor neurons, sensory neurons, interneurons, or projection neurons of the subject.

[0019] Therefore, the present invention makes it possible to create a virus that accesses these neurons after intracranial or intraspinal intramuscular injection, and subsequently modifies gene expression in these neurons, for the purpose of curing, alleviating symptoms, and / or improving the quality of life of patients with diseases affecting these neurons.

[0020] As discussed above, AAV capsids have the potential for exquisite levels of cytotropy and cephalotropy, but currently available screening technologies cannot utilize this selectivity and thus cannot contribute to therapeutic efficacy.

[0021] The present invention overcomes these drawbacks and enables screening of AAV capsid libraries in human cells derived from induced pluripotent stem cells ("iPSCs") or embryonic stem cells ("ESCs"). Human cells, including many subtypes of neurons, can be induced in vitro from iPSCs or ESCs (Little et al., 2019). Since the genetic makeup of these neurons may be more similar to that of human patients than to that of the same cells found in animal neurons, they are considered to provide a much more suitable substrate for screening AAV libraries than experimental animals. In some embodiments, the neurons are motor neurons. Motor neurons can be derived from neurons in human embryos, but such neurons are limited in supply and not individualized. In contrast, inducing neurons from iPSCs, as taught by some embodiments of the present invention, makes it possible to supply a greater number of motor neurons that are also individualized.

[0022] The present invention also includes a viral evolution approach that uses a combination of induced iPSCs and ESC-derived neurons and in vitro screening to identify nucleotide sequences encoding AAV capsids that allow AAV capsids to more efficiently infect neurons via injection. In some embodiments, this approach enables the identification of nucleotide sequences encoding AAV capsids that allow AAV capsids to more efficiently infect motor neurons via intramuscular injection.

[0023] The present invention also includes a technology platform for identifying novel adeno-associated virus (AAV) capsids based on the ability to target individual compartments (or sub-compartments) of neurons by combining the use of iPSC / ESC-derived cell cultures and microfluidic chambers. Examples of its use to screen AAV capsid libraries for the ability to efficiently infect the terminals of motor neurons are described herein, but the same system may be used to identify capsids targeted to many neuron types.

[0024] Furthermore, since the capsid sequences that efficiently infect neurons can vary among subjects, the present invention further includes a process of collecting skin samples from individual subjects, converting those skin samples into stem cells and then into neurons, and screening for effective AAV vectors using these neurons from the patient to provide an individualized approach to gene therapy (see Figure 2).

[0025] This invention is more beneficial than other treatments because it may only require a single procedure. This screening method makes it possible to discover novel capsids so that patients are not immune to the viral particles containing them. In the case of motor neurons, rAAV is thought to be deliverable to any muscle (i.e., even muscles that are difficult to access under anesthesia in the operating room). Since the injection can be applied only to specific symptomatic muscles, the capsid may have low off-target effects and complications. At the same time, it is possible to repeatedly deliver different capsids to the patient if necessary (e.g., to different muscles) without causing immune-mediated rejection.

[0026] The present invention may also be applicable to other motor neuron disorders for which there are currently few or no effective treatments, as well as to other types of neurons underlying the neurological disorders.

[0027] The rAAV vectors developed using the methods described herein are particularly applicable to the treatment of neuromuscular / neuromotor disorders such as spasticity, amyotrophic lateral sclerosis, and dystonia, enabling the introduction of genetic material into motor neurons by intramuscular injection of the viral vector. For example, this specification discloses how the methods described herein can form the basis of gene therapy aimed at alleviating symptoms of spasticity.

[0028] However, the screening methods defined herein can be used to screen capsid sequences that effectively target many neuron types, regardless of the disease being treated, and therefore may be effective in gene therapy approaches regardless of neuron disorders (or "neuronal disorders"). To the best of our knowledge, no methods / inventions suggesting screening AAV libraries in stem cell-derived cells or constructing personalized AAV vectors for gene therapy are currently documented in the literature.

[0029] Next, we will discuss in more detail some specific aspects of the present invention.

[0030] A method for screening nucleotide sequences that encode the capsid of adeno-associated virus ("AAV") particles capable of infecting neurons in a test subject. In one embodiment, the present invention provides a method for screening nucleotide sequences encoding the capsid of adeno-associated virus ("AAV") particles capable of infecting neurons in a subject, the method being: (i) A step of providing a population of neurons, wherein the neurons are derived from induced pluripotent stem cells ("iPSCs") or embryonic stem cells ("ESCs"), (ii) The step of bringing the group into contact with a first set of (or “library”) test AAV particles, (iii) the step of isolating a first group of AAV particles that have infected those neurons, and (iv) The step of determining the nucleotide sequences that encode the capsids of the first group of AAV particles that have infected those neurons. Includes.

[0031] In another embodiment, the present invention provides a method for screening nucleotide sequences encoding the capsid of adeno-associated virus ("AAV") particles capable of infecting specific subcompartments of neurons in a subject, the method being: (i) A step of providing a population of neurons, wherein the neurons are derived from induced pluripotent stem cells ("iPSCs") or embryonic stem cells ("ESCs"); (ii) The step of bringing the group into contact with a first set of (or “library”) test AAV particles, (iii) the step of isolating a first group of AAV particles that have infected specific subcompartments of those neurons, and (iv) The step of determining the nucleotide sequences that encode the capsids of the first group of AAV particles that have infected those neurons. Includes.

[0032] In another embodiment, the present invention provides a method for screening nucleotide sequences encoding the capsid of adeno-associated virus ("AAV") particles capable of infecting neurons in a subject, the method being: (i) A step of providing a population including neurons, wherein the neurons are derived from induced pluripotent stem cells ("iPSCs") or embryonic stem cells ("ESCs"), and each neuron has a first specific subcompartment and a second specific subcompartment, (ii) The process of arranging neurons such that a first specific subcompartment and a second specific subcompartment are far apart from each other. (iii) The step of bringing a first specific subcompartment into contact with a first multiple (or “library”) of test AAV particles, (iv) A step of isolating a first group of AAV particles that have infected a second specific subcompartment, and (v) The process of determining the nucleotide sequences encoding the capsids of the first group of AAV particles that have infected a second specific subcompartment of a neuron. Includes.

[0033] In this method, the second specific subcompartment is the “target” subcompartment, which in some preferred embodiments is the cell body of a neuron. In some embodiments, the population of neurons is a neuronal enrichment population, where 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99%, or 100%, of all cells in that population are neurons. In some embodiments, the nucleotide sequence encoding the capsid determined in the screening method disclosed herein may be used for a further number of directional evolutionary steps (viral evolutionary approach) to increase evolutionary pressure. Thus, in some embodiments, after determining the nucleotide sequence encoding the capsid of a first plurality of AAV particles that have infected a neuron or a specific subcompartment of a neuron, (i) A step of preparing a second set of test AAV particles (or a “library”) using nucleotide sequences encoding the capsids of a first set of AAV particles. (ii) Repeating the steps of the screening method to isolate a second set of AAV particles that have infected a neuron or a specific subcompartment of a neuron, and (iii) The step of determining the nucleotide sequences encoding the capsids of a second group of AAV particles that have infected a neuron or a specific subcompartment of a neuron. It further includes, The nucleotide sequences encoding the capsids of the second set of AAV particles are more effective at infecting neurons or specific subcompartments of neurons than the nucleotide sequences encoding the capsids of the first set of AAV particles.

[0034] The effectiveness of AAV particles infecting neurons or specific subcompartments of neurons can be determined by counting the number of neurons expressing viral DNA. In some cases, multiple motor neurons may innervate the same target, for example, muscles in the case of motor neurons, and the proportion of these neurons transduced by AAV particles can be counted. The effectiveness of AAV particles infecting neurons or specific subcompartments of neurons can also be determined by DNA sequencing or RT-PCR to examine the "copy number" of viral DNA present in nerve cells. This will allow for an estimate of how many times the same cell has been infected with AAV particles.

[0035] In some embodiments, a second set of test AAV particles are i) Random mutagenesis of the nucleotide sequences encoding the capsids of the first set of AAV particles, ii) Shuffling of the nucleotide sequences encoding the capsids of the first plurality of AAV particles, and iii) Insertion of a targeted peptide sequence or random peptide sequence up to 25 amino acids in length into various regions of VP1, VP2, or VP3 of the nucleotide sequence encoding the capsid of the first plurality of AAV particles. It is made by one or more of the following:

[0036] In some cases, the neuron subcompartment is the cell body, neurite, axon, or dendrite. In some cases, the neuron subcompartment is the axon terminal (or "synaptic terminal"). In some cases, the second subcompartment is the cell body. In some cases in step (iii) above, a targeted peptide sequence or random peptide sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids is inserted into various regions of VP1, VP2, or VP3 of the nucleotide sequence encoding the capsid of the first plurality of AAV particles. In some cases, these additional steps may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0037] In embodiments of the present invention in which the subcompartments of a neuron are far apart from each other, the neuron is cultured in such a manner that the cell body and neurite can be separated so that the rAAV library can be applied to one and not to the other (for example, by using a microfluidic chamber that separates the cell body, axon and synaptic terminal of the neuron for the purpose of screening the library).

[0038] In embodiments of the present invention in which the subcompartments of a neuron are far apart from each other, a first specific subcompartment and a second specific subcompartment may be grouped in different physical regions of one or more containers. The first specific subcompartment and the second specific subcompartment may be connected by axons. In some cases, the first specific subcompartment is a synaptic terminal of a nerve, and the second specific subcompartment is a nerve cell body.

[0039] In embodiments of the present invention in which neuronal subcompartments are far apart from each other, a first specific subcompartment and a second specific subcompartment may be grouped in different physical regions of one or more containers within a microfluidic chamber. These one or more containers may be further separated by microfluidic channels. Neurons may grow further once added to the microfluidic chamber. The microfluidic chamber may be an AXIS® Axon Isolation Device.

[0040] By applying an AAV capsid library to one component of a neuron (e.g., axon, dendrite, neurite, or axon terminal (or "synaptic terminal")) and collecting genetic material from another region of the cell (e.g., the cell body), it becomes possible to identify and determine the AAV capsid sequences that successfully infected those cells via that specific pathway.

[0041] There are several variations of libraries that can be used as a starting point, and these can be evaluated by determining gene sequences from AAVs and comparing them with AAVs from different libraries. Furthermore, the time between i) exposing neurons to a library of AAVs and ii) collecting neurons to isolate the AAVs that have infected them ("incubation time") can be optimized.

[0042] In some embodiments, multiple types of cells derived from iPSCs or ESCs may be added to a population of neurons in order to evolve AAV capsids toward or against infection of one or more of those cell types. By collecting genetic material from all or some of these cells individually (e.g., motor neurons only) or in combination (e.g., motor neurons and sensory neurons), it becomes possible to determine the capsid sequences that successfully infect these cells.

[0043] In some embodiments, such additional cells may be skeletal muscle cells, muscle cells, or sensory neurons. The additional cells may be myotubes. The additional cells may also be myoblasts or muscle fibers. The additional cells may be C2C12 cells.

[0044] In some cases, iPS cells or ESCs are derived from the subject. In some cases, iPSCs or ESCs are derived from the subject's skin sample. In some cases, iPSCs or ESCs are derived from the subject's fibroblasts. In some cases, the subject is a human subject. iPSCs and ESCs may be obtained from animals, human subjects / patients, or cell banks. Screening an AAV capsid library using iPS cells from a human subject or human patient makes it possible to identify capsid sequences that infect neurons or other cells of that subject or patient. Therefore, using this method, capsid sequences can be produced on an individualized basis.

[0045] In some embodiments, step (i) includes the step of inducing neurons from i) iPSC or ii) ESC.

[0046] In some cases, the above method is a method for screening nucleotide sequences that encode the capsid of adeno-associated virus ("AAV") particles that can infect neurons in a subject via intramuscular injection.

[0047] In some cases, neurons are derived from iPS cells. In some cases, neurons are derived from ESCs. In some cases, human iPSCs may be easier to retrieve than ESCs.

[0048] As used herein, the term “neuron” includes a neuron and parts thereof (e.g., the neuronal cell body, axon, or dendrites). As used herein, the term “neuron” refers to a nervous system cell that includes the central cell body or soma and two types of extensions or projections: dendrites, which generally transmit most of the neuronal signal to the cell body, and axons, which generally transmit most of the neuronal signal from the cell body to effector cells (e.g., target neurons or muscles). Neurons can transmit information from tissues and organs to the central nervous system (afferent neurons or sensory neurons) and can transmit signals from the central nervous system to effector cells (efferent neurons or motor neurons). Other neurons called interneurons connect neurons within the central nervous system (brain and spinal cord). Other neurons called projection neurons extend their axons from one area of ​​the nervous system to another. The screening methods disclosed herein may be used to screen for nucleotide sequences encoding the capsids of adeno-associated virus ("AAV") particles capable of infecting such neurons. In some preferred embodiments, the neurons are motor neurons. In other preferred embodiments, the neurons are sensory neurons, interneurons, or projection neurons.

[0049] Furthermore, as mentioned above, the screening methods described above could be applied to screen for nucleotide sequences encoding the capsids of AAV particles capable of infecting other cell types (e.g., sensory neurons, basal ganglia projection neurons, dopaminergic neurons, and muscle tissue).

[0050] In some cases, the screening methods described herein include the step of providing a number of test AAV particles that have been additionally screened for their ability to infect muscle cells, for example, by direct intramuscular injection. The additional screening may be performed before, after, or concurrently with screening the particles for their ability to infect neurons.

[0051] The methods described herein may be in vivo or in vitro.

[0052] Culture of fibroblasts from human skin biopsy material In some embodiments, the iPSCs used in the screening methods described herein are derived from human test subjects. In some embodiments, the iPSCs are derived from fibroblast cultures from skin biopsy material of the subject.

[0053] Several methods have been previously reported that enable the culture of fibroblasts from skin biopsy material (Vangipuram M, Ting D, Kim S, Diaz R, Schule B. Skin punch biopsy explant culture for derivation of primary human fibroblasts. J Vis Exp. 2013;(77):e3779. Published July 7, 2013 doi:10.3791 / 3779).

[0054] In some preferred embodiments, the above method includes one or more of the following steps. - Under sterile conditions, place the skin biopsy material into a gelatin-coated culture dish containing Dulbecco's modified Eagle medium (DMEM) with 20% fetal bovine serum (FBS). - Under a surgical microscope, cut the biopsy material into pieces approximately 200-500 μm in size. Transfer 2-3 pieces of biopsy material to a new gelatin-coated culture dish containing DMEM / 20% FBS. - Change the culture medium every two days until the fibroblasts reach confluence. The cells can then be trypsinized and transferred to a new culture vessel for expansion. The cells can be frozen in liquid nitrogen or used for induction into IPSCs.

[0055] Production of iPSCs from fibroblasts In some embodiments, the iPSCs used in the screening methods described herein are derived from human test subjects. In some embodiments, the iPSCs are derived from fibroblast cultures from skin biopsy material of the subject.

[0056] Induction of human fibroblasts into IPSCs can be achieved using commercially available kits, such as the ThermoFisher CytoTune-IPS Sendai Reprogramming Kit. Further details can be found at https: / / www.thermofisher.com / order / catalog / product / A16517# / A16517 and https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / cytotune_ips_2_0_sendai_reprog_kit_man.pdf.

[0057] This involves introducing Yamanaka factors (Oct3 / 4, Sox2, Klf4, c-Myc) into somatic cells using Sendai virus. These factors are highly expressed in embryonic stem cells, and overexpression induces pluripotency in human and mouse somatic cells (see Takahashi, K, Yamanaka, S. A decade of transcription factor-mediated reprogramming to pluripotency. Nat Rev Mol Cell Biol 17, 183-193 (2016). https: / / doi.org / 10.1038 / nrm.2016.8). Several methods are available that enable the creation of IPSCs from fibroblasts, and these methods may depend on introducing some combination of these factors into fibroblasts.

[0058] In some preferred embodiments, a method for producing iPSCs from fibroblasts includes one or more of the following steps: -Plate the fibroblasts to achieve 30-60% confluence. - Complete the introduction of each factor at the following MOI (Minimum Infection) levels: KOS MOI=5, hc-Myc MOI=5, hKlf4 MOI=3). -Apply an appropriate volume of each Sendai virus to fibroblasts (day 0), and incubate the cells overnight at 37°C / 5% CO2. -The culture medium is changed on the following day (day 1), and then every two days thereafter until day 7. -On day 7, the cells are trypsin-treated, counted, and replated onto a 6-well plate coated with vitronectin at a density of approximately 2 × 10^5 cells per well. -On day 8, change the culture medium to Essential 8 medium and change it daily thereafter. Three to four weeks after the initial transduction, the IPSC colonies should have grown to an appropriate size. - IPSC colonies are collected manually or using chemical dissociation and stored in liquid nitrogen, or used directly for induction into primary cell types. -iPSCs are maintained on Geltrex (Life Technologies) using Essential 8 Medium (Life Technologies) and subcultured at 37°C and 5% carbon dioxide using EDTA (Life Technologies, 0.5 mM). In some cases, the use of Geltrex may increase cell viability compared to other substrates. In some cases, the use of EDTA and Essential 8 medium is particularly useful for producing human iPSCs, with EDTA increasing cell viability by approximately 20% compared to other dissociation methods.

[0059] Differentiation of iPSCs into motor neurons that innervate limbs In some embodiments, the iPSCs and ESCs used in the above method are differentiated into somatic motor neurons.

[0060] Several methods that enable the differentiation of iPSCs or ESCs into somatic motor neurons have been previously described below. Wichterle, H. and Peljto, M. (2008),Differentiation of Mouse Embryonic Stem Cells to Spinal Motor Neurons.Current Protocols in Stem Cell Biology,5:1H.1.1-1H.1.9.doi:10.1002 / 9780470151808.sc01h01s5; Journal of Neuroscience 8 September 2004,24(36)7848-7858;DOI:10.1523 / JNEUROSCI.1972-04.2004;and Amoroso MW, Croft GF, Williams DJ, et al. Accelerated high-yield generation of limb-innervating motor neurons from human stem cells. J Neurosci.2013;33(2):574-586.doi:10.1523 / JNEUROSCI.0906-12.2013.

[0061] In some preferred embodiments, a method for enabling the differentiation of iPSCs into motor neurons innervating a limb includes one or more of the following steps: -IPSCs are maintained in suspension in a medium consisting of DMEM:nutrient mixture F-12 (DMEM / F:12;Invitrogen), which contains 20% Knockout Serum Replacer (Invitrogen), 110 μM β-mercaptoethanol, l-glutamine and non-essential amino acids (NEAA;Invitrogen), and 20 ng / ml basic fibroblast growth factor (bFGF;Invitrogen), supplemented with 10 μM Rho-related kinase inhibitor Y27632 (Ascent Scientific) to enhance single-cell viability, 20 ng / ml bFGF (Invitrogen) to enhance growth, and 10 μm SB435142 (SB;Sigma) and 0.2 μM LDN193189 (LDN;Stemgent), Neurobasal, N2 supplement, B27 supplement, and insulin for neurogenesis. Cells are subcultured using dispase. In some cases, cell viability can be improved by using Neurobasal, N2 supplements, B27 supplements, insulin, and / or dipase. On day 0, switch embryoid bodies (EBs) to neuroinduction medium (l-glutamine; NEAA; penicillin / streptomycin; heparin, 2 μg / ml; DMEM / F:12 with N2 supplement; Invitrogen) and 1 μM dorsomorphin (Millipore), 2 μM SB431542 (Tocris Bioscience), and 3 μM CHIR99021 (Miltenyi Biotec). On day 2, add all-trans retinoic acid (RA; 1 μm; Sigma), ascorbic acid (0.4 μg / ml; Sigma), and brain-derived neurotrophic factor (10 ng / ml; R&D). In some cases, cell viability is improved by using 1 μM dorsomorphin, 2 μM SB431542, and / or 3 μM CHIR99021. -On day 8, the culture was enzymatically dissociated using dispase (GIBCO, 1 mg / ml), plated onto a laminin-coated plate, and then patterned for 7 days with 1 μM retinoic acid (Sigma), ascorbic acid (0.4 μg / ml; Sigma), and brain-derived neurotrophic factor (10 ng / ml; R&D). In some cases, this step improves cell viability. -On day 14, spinal cord MN precursors were treated with 0.1 μM palmorfamine for an additional 4 days. In some cases, this step improves cell viability. -On day 18, the basal medium is changed to Neurobasal (Invitrogen), which contains all the previously used factors, plus 10 ng / ml each of insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) (R&D), as well as B27 (Invitrogen) and 0.1 μM compound E (Enzo Life Sciences). In some cases, the use of 0.1 μM compound E improves cell viability. -Approximately 5 days later, EB is dissociated with 0.05% trypsin (Invitrogen) and plated onto polylysine / laminin-coated coverslips, or directly plated into the motor neuron compartment of a microfluidic chamber for use in AAV screening (see Figure 3).

[0062] Differentiation of iPSCs into skeletal muscle fibers In some preferred embodiments, iPSCs may also be induced into skeletal muscle cells (or "skeletal muscle fibers"), which may be added to the population of motor neurons used in the screening methods described herein.

[0063] Several methods for differentiating iPSCs into muscle fibers have been previously described below. Maffioletti, S., Gerli, M., Ragazzi, M. et al. Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells. Nat Protoc 10,941-958(2015). https: / / doi.org / 10.1038 / nprot.2015.057.

[0064] In some preferred embodiments, a method for differentiating iPSCs into muscle fibers includes one or more of the following steps: - Dissociated IPSC colonies are cultured on Matrigel-coated dishes in MEM-α (Minimal Essential Media alpha) medium containing 10% fetal bovine serum, 2 mM glutamine, and 0.1 mM 2-mercaptoethanol. - Replace the culture medium daily for one week. -Once 100% confluence is achieved, detach the cells from Matrigel using trypsin, at a rate of 1.2 × 10^4 cells / cm². 2 Re-plating onto plastic at that density. - The cells can be enlarged and cryopreserved for future use, or they can be used for muscle differentiation. - For induction, cells are plated in 35 mm dishes at a density of approximately 1 × 10^5 in a medium containing MegaCell DMEM with 5% fetal bovine serum, 2 mM glutamine, 1% essential amino acids, 50 μM 2-mercaptoethanol, and 5 ng / ml basic fibroblast growth factor. - Lentiviruses expressing myoblast-determining protein 1 (MDP1) are used to infect cultures with infection efficiencies of 1, 10, and 50 in the presence of polybren. - Examine the cells daily and enlarge the culture as needed. -Myotubes typically become identifiable about 3 days after MDP1 expression. A portion of the culture can be fixed and tested for myosin heavy chain expression. - Re-plat the remaining culture into the microfluidic chamber of the muscle compartment as needed (see Figure 3). These muscle cells are typically cultured in the muscle compartment about 5–7 days after the addition of motor neurons (by which point the axons of the motor neurons should have begun to enter the muscle compartment). - By maintaining the culture for approximately 1-3 weeks as needed, it becomes possible to add the AAV capsid library.

[0065] Fabrication of AAV libraries and application of AAV capsid libraries to neurons in a microfluidic chamber. A variety of capsid libraries can be created through processes such as i) random mutagenesis of naturally occurring capsids, ii) shuffling of naturally occurring capsids, iii) insertion of targeted peptide sequences or random peptide sequences up to 25 amino acids in length into various regions of VP1, VP2, or VP3 of AAV capsids, or iv) a combination of the above processes.

[0066] To construct the above library, randomized capsid sequences are cloned into an AAV backbone containing the AAV2 terminal inversion sequence (ITR; packaging signal) and the AAV rep gene. These DNA plasmids are transfected into HEK293 cells in the presence of additional adenovirus genes to facilitate AAV packaging. AAV virions are collected from HEK293 cells and / or culture medium and purified and concentrated according to standard methods (e.g., Potter et al., 2014 https: / / dx.doi.org / 10.1038%2Fmtm.2014.34; McClure et al., 2011 http: / / dx.doi.org / 10.3791 / 3348).

[0067] In some embodiments, a method for creating an AAV library includes one or more of the following steps: - The purified and concentrated AAV library is diluted in Dulbecco's modified Eagle medium (DMEM) and applied to the muscle chamber of a microfluidic device. - Two to ten days after application, nerve cell bodies are collected by chemical methods (i.e., trypsin treatment) or mechanical methods (cell scraping). - Neurons can be lysed, and the lysate product can be subjected to deep sequencing (e.g., RNA-seq) to directly detect the capsid sequence found in the neurons, or the lysate product can be used as a PCR template with primers targeting the conserved region of the AAV capsid. After PCR of the capsid region, the DNA fragment is cloned into a DNA vector and subjected to Sanger sequencing. - By analyzing the conserved regions of capsid sequences collected from neurons using bioinformatics, synthesizing highly enriched capsids (de novo synthesis), and then repeating further mutagenesis or in vitro screening, evolutionary pressure due to directional evolution can be increased. - Directional evolution can be repeated several times (approximately 2-5 times). Capsid sequences that exhibit efficient retrograde transport in vitro can be used to create functional virions for in vivo use in animals or humans.

[0068] rAAV vector containing a capsid sequence that can lead to neuronal infection In one embodiment, the present invention provides an AAV capsid identified by the screening method of the present invention. In another embodiment, the present invention provides a nucleotide sequence encoding an AAV capsid having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the present invention provides a nucleotide sequence encoding an AAV capsid having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 11. In another embodiment, the present invention provides nucleotide sequences encoding AAV capsids having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to SEQ ID NO: 13.

[0069] In some cases, the nucleotide sequences disclosed herein differ from the wild-type AAV vector by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 base pairs.

[0070] In some cases, the nucleotide sequences disclosed herein differ from SEQ ID NO: 13 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 base pairs. In some cases, the nucleotide sequences disclosed herein differ from SEQ ID NO: 13 by 1-10, 2-8, or 4-6 base pairs.

[0071] Nucleotides can be used in the development of gene therapies that involve viruses that access neurons after intramuscular injection and subsequently modify neuronal activity and / or gene expression.

[0072] Accordingly, in another embodiment, the present invention provides an adeno-associated virus ("AAV") expression vector comprising a capsid nucleotide sequence of an AAV capsid identified by the screening method of the present invention. In another embodiment, the present invention provides a recombinant adeno-associated virus ("AAV") expression vector comprising a nucleotide sequence encoding a capsid having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the present invention provides a recombinant adeno-associated virus ("AAV") expression vector comprising a nucleotide sequence encoding a capsid having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to SEQ ID NO: 11. In another embodiment, the present invention provides a recombinant adeno-associated virus ("AAV") expression vector comprising a nucleotide sequence encoding a capsid having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to SEQ ID NO: 13.

[0073] In some embodiments, the expression vector can alter the activity of targeted motor neurons in a subject. In some embodiments, the expression vector can alter the activity of targeted motor neurons in a subject via intramuscular injection. In some cases, the expression vector may further include a nucleotide sequence encoding a second capsid that can prevent an immune response from being triggered in the subject.

[0074] Expression vector As used herein, an expression vector is a DNA molecule used to introduce and express foreign genetic material within a cell. Such a vector contains a promoter sequence operably ligated to a gene encoding the protein to be expressed. "Promoter" means the minimum DNA sequence sufficient to direct the transcription of the DNA sequence to which it is operably ligated. "Promoter" also means containing sufficient promoter elements for controllable promoter-dependent gene expression for cell-type specific expression. Such elements may be located in the 5' or 3' region of the native gene. Alternatively, the expression vector may be an RNA molecule that undergoes reverse transcription into DNA as a result of reverse transcriptase.

[0075] The expression vector may include a stop codon and an expression enhancer. Any suitable vector, enhancer, and stop codon can be used to express a gene product such as a Kv1 potassium channel from the expression vector according to the present invention. The expression vector includes viral vectors such as AAV vectors.

[0076] Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. In addition to the elements of the present invention described above, suitable vectors can be selected or constructed that appropriately include appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, marker genes, and other sequences. Molecular biological methods suitable for polypeptide expression in cells are well known in this art. For further details, see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons (1995 and periodic supplements).

[0077] As used herein, the term “operatably linked” includes a situation in which a selected gene and a promoter are covalently linked so as to place the gene’s expression (i.e., the polypeptide encoding) under the influence or control of the promoter. Therefore, a promoter is operatably linked to a gene if it can transcribe the gene into RNA within a cell. If necessary, the resulting RNA transcript can then be translated into the desired protein or polypeptide. The promoter is suitable for expressing an operatably linked gene in mammalian cells. Preferably, the mammalian cell is a human cell.

[0078] AAV Vector The vector is a recombinant AAV vector. AAV vectors are relatively small DNA viruses that can be stably and site-specifically integrated into the genome of infected cells. AAV vectors can infect a wide range of cells without inducing significant effects on cell growth, morphology, or differentiation. The AAV genome has been cloned, sequenced, and characterized. The AAV genome consists of approximately 4700 base pairs and includes approximately 145 base pairs terminal inversion (ITR) regions at each end that act as replication origins for the virus. The remainder of the genome is divided into two essential regions responsible for capsid formation: the left side of the genome containing the rep gene, which is involved in viral replication and viral gene expression, and the right side of the genome containing the cap gene, which codes for the viral capsid protein.

[0079] AAV vectors can be prepared using standard methods in this field. Any serotype of adeno-associated virus is preferred (e.g., Blacklow, “Parvoviruses and Human Disease” JRPattison, ed. (1988) pp. 165-174; Rose, Comprehensive Virology 3:1, 1974; P. Tattersall “The Evolution of Parvovirus Taxonomy” in Parvoviruses (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) pp. 5-14, Hudder Arnold, London, UK (2006); and DE Bowles, JE Rabinowitz, RJ Samulski “The Genus Dependovirus” (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) pp. 15-23, Hudder Arnold, London, UK (2006) (these disclosures are incorporated herein by reference in their entirety). Methods for purifying vectors can be found, for example, in U.S. Patents 6,566,118, 6,989,264 and 6,995006, and in International Patent Application Publication No. W0 / 1999 / 011764 entitled “Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors” (these disclosures are incorporated herein by reference in their entirety).

[0080] The preparation of hybrid vectors is described, for example, in PCT application number PCT / US2005 / 027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for gene transfer in vitro and in vivo is described (see, for example, International Patent Application Publications WO1 / 18088 and WO93 / 09239, U.S. Patents 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced with the gene of interest, and the use of these constructs for the transfer of the gene of interest in vitro (into cultured cells) or in vivo (directly into living organisms). The replication-deficient recombinant AAV according to the present invention can be prepared by cotransfecting a cell line infected with a human helper virus (e.g., adenovirus) with a plasmid containing the target nucleic acid sequence adjacent to two AAV terminal inversion (ITR) regions, and a plasmid containing AAV capsid-forming genes (rep and cap genes). The resulting recombinant AAV is then purified by standard methods.

[0081] In some embodiments, AAV vectors useful for expression constructs as described herein include those encapsulated in a capsid to form viral particles (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, among others). Accordingly, this disclosure includes recombinant viral particles (recombinant, since they contain recombinant polynucleotides) comprising any of the vectors described herein. In some preferred embodiments, the AAV viral particle is AAV2. In some preferred embodiments, the AAV viral particle is AAV6. The use of AAV6 may improve the effectiveness of motor neuron infection after intramuscular injection.

[0082] In some embodiments, the viral vector includes a sequence encoding a reporter protein, such as a fluorescent protein. In other embodiments, the viral vector lacks a sequence encoding a reporter protein, such as a fluorescent protein.

[0083] In some embodiments, the viral vector further includes genes encoding viral packaging proteins and envelope proteins.

[0084] The expression vector may further contain a rep gene, which, if necessary, is the AAV2rep gene. The expression vector may further contain a cap gene, which, if necessary, is the AAV2cap gene. The expression vector may further contain a terminal inversion sequence, which, if necessary, is the AAV2 terminal inversion sequence. The expression vector may further contain a gene encoding the viral packaging protein and / or envelope protein. The expression vector may further contain a regulatory gene, which, if necessary, is poly(A).

[0085] AAV vector containing further transgenes In some embodiments, the expression vector further comprises a transgene encoding a transgene product that can alter the activity of targeted motor neurons in a subject. In some embodiments, the transgene product can alter the activity of targeted motor neurons in a subject via intramuscular injection.

[0086] In some embodiments, the gene product can alter the excitability of neurons when the expression vector is administered to a subject. In some embodiments, the gene product can reduce the hyperexcitability of neurons when the expression vector is administered to a subject. The transgene held by the expression vector depends on the patient's symptoms. For example, in the case of spasticity, the payload may aim to alleviate the hyperexcitability of motor neurons. In some embodiments, the transgene expressed by a virus alters the electrical properties of neurons. This may involve the use of a specific promoter to initially drive one of several different transgenes, i.e., one that simply reduces neuronal excitability (e.g., KCC2, Kv1), or one that completely blocks synaptic transmission (e.g., tetanus toxin light chain), or one that responds to a low dose of medication taken orally by the patient so that symptoms can be managed by dose adjustment (e.g., responsive to uPSEM or varenicline, responsive to clozapine PSAM4-GlyR, responsive to DREADD hM4Di). To the best of our knowledge, there are currently no literature descriptions suggesting a combination of intramuscular gene delivery and altering neuronal excitability to treat spasticity.

[0087] DREADD hM4Di is a mutated muscarinic acetylcholine receptor that can bind to synthetic ligands such as clozapine-N-oxide to deactivate neuronal activity. KCC2 is a potassium-chloride transporter found in neurons that can push chloride ions out of cells and controls neuronal excitability. Tetanus toxin light chain is part of the tetanus toxin, a neurotoxin that can specifically cleave a protein (VAMP2) on synaptic vesicles. Cleavage of VAMP2 prevents synaptic vesicle docking and neurotransmitter release. Potassium channels such as Kv1 can specifically reduce neuronal excitability. In some cases, the ability of a gene product to alter the excitability of a motor neuron can be measured by electrophysiological recording (patch-clamp) in infected cells. The response of an infected neuron to an injected current (frequency of the action potential), measured as the slope of the frequency / current (f / I) curve, can be recorded to measure its excitability. This may include dose-response curves for the ligands listed above.

[0088] Once the expression vector is administered to a subject, the transgene product may be able to reduce neuronal excitability. In some embodiments, the transgene or transgene product is the KCC2 transgene or KCC2 transgene product. Potassium chloride transporter member 5 (KCC2) is a neuron-specific potassium chloride cotransporter involved in establishing a chloride ion gradient within neurons by maintaining low intracellular chloride concentrations. Animals with reduced expression of this transporter exhibit severe motor impairment, epileptic-like activity, and spasticity. In some embodiments, the KCC2 transgene has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to sequence number 3, or the KCC2 transgene product has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to sequence number 4.

[0089] In some embodiments, the transgene or transgene product is the Kv1 transgene or Kv1 transgene product. The Kv1 potassium channel is a voltage-gated, delayed-rectifying potassium channel phylogenetically related to the Drosophila Shaker channel. Voltage-gated potassium channels regulate excitability by opening and closing potassium-selective pores in response to electrical potential. Often, when intracellular particles block the pores, the flow of potassium ions can be obstructed, a process known as rapid inactivation. The subunits of the Kv1 potassium channel have six putative transmembrane segments, and loops between the fifth and sixth segments of each of the four Kv1 subunits that constitute the complete channel form the pore. In some embodiments, the Kv1 transgene has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to sequence number 5, or the Kv1 transgene product has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with respect to sequence number 6.

[0090] The transgene product may be capable of blocking synaptic transmission in neurons when the expression vector is administered to a subject. In some embodiments, the transgene or transgene product is a tetanus toxin light chain transgene or a tetanus toxin light chain transgene product. In some embodiments, the tetanus toxin light chain transgene has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 7, or the tetanus toxin light chain transgene product has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 8.

[0091] The transgene product may be a receptor activated solely by a synthetic ligand (RASSL) or a designer receptor activated exclusively by a designer drug (DREADD). RASSL and DREADD are a class of chemogenetically engineered proteins that enable spatial and temporal control of G protein signaling in vivo. In some embodiments, the transgene or transgene product is an hM4Di transgene or an hM4Di transgene product. In some embodiments, the hM4Di transgene has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 7, or the hM4Di transgene product has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 8.

[0092] In some preferred embodiments, the transgene is operably linked to a neuron-specific promoter.

[0093] The calculation of amino acid sequence or nucleotide sequence alignment and identity percentage can be achieved in various ways known to those skilled in the art using publicly available computer software such as ClustalW 1.82, T-coffee, or Megalign (DNASTAR) software. When using such software, it is preferable to use default parameters such as gap penalties and elongation penalties. The default parameters for ClustalW 1.82 are protein gap open penalty = 10.0, protein gap elongation penalty = 0.2, protein matrix = Gonnet, protein / DNA ENDGAP = -1, and protein / DNA GAPDIST = 4.

[0094] Next, (N / T) *The identity percentage can be calculated from multiple alignments, with N being 100. Here, N is the number of positions where the two sequences share the same residue, and T is the total number of positions being compared. Alternatively, the identity percentage is (N / S) * It can be calculated as 100, where S is the length of the shorter sequence being compared. The amino acid / polypeptide / nucleic acid sequence may be newly synthesized (de novo synthesis), or it may be a naturally occurring amino acid / polypeptide / nucleic acid sequence or a derivative thereof.

[0095] It is clear that, due to the degeneracy of the genetic code, any nucleic acid sequence can be modified or altered without substantially affecting the sequence of the protein it encodes, thereby generating functional variants. Preferred nucleotide variants have sequences altered by the substitution of different codons that code for the same amino acid within their sequence, thus resulting in silent alterations. Other preferred variants have homologous nucleotide sequences but include all or part of a sequence altered by the substitution of different codons that code for amino acids having side chains with similar biophysical properties to the replacing amino acid, in order to result in a conservation change. Examples of small, nonpolar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Examples of large, nonpolar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Examples of polar, neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Examples of positively charged (basic) amino acids include lysine, arginine, and histidine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0096] Addition of transgene products to alter neuronal activity For testing various AAV transgene products (or "cargo" or "payload") that may be used to alter neuronal activity, genes can be cloned into an AAV scaffold containing an AAV terminal inversion sequence (ITR) along with a general or cell-type targeted promoter and conventional regulatory elements (e.g., the Woodchuck posttranslational regulatory element and polyA).

[0097] The genes used depend on the patient's needs and may belong to one of two classes: (a) ligand-independent, which sustainably reduces excitation, or (b) ligand-dependent, which can modulate neuronal excitability by taking an approved drug.

[0098] Within each category, several genes may be tested on the patient's neurons using electrophysiological methods to determine the best choice for that patient.

[0099] In some preferred embodiments, a method for adding cargo to alter neuronal activity may include one or more of the following steps: -The capsid sequence identified in the screening method described herein is newly synthesized (de novo synthesis) and inserted into an AAV helper plasmid (Rep / Cap) containing the AAV2 REP gene. - The Rep / Cap plasmid is combined with an additional plasmid containing the AAV backbone and an adenovirus helper gene (e.g., pHelper), and transiently transfected into HEK293 cells. - AAV particles can be purified using standard methods and used in in vitro or in vivo experiments (e.g., Potter et al., 2014 https: / / dx.doi.org / 10.1038%2Fmtm.2014.34; McClure et al., 2011 http: / / dx.doi.org / 10.3791 / 3348).

[0100] Virus particles The present invention also includes an in vitro method for producing AAV virus particles. In one embodiment, the method comprises transducing a viral vector or expression vector, as described herein, into mammalian cells, expressing the viral packaging protein and envelope protein necessary for particle formation within those cells, and culturing the transduced cells in a culture medium so that the cells produce viral particles to be released into the culture medium. A suitable example of mammalian cells is human embryonic kidney (HEK) 293 cells.

[0101] It is possible to use a single expression vector that encodes all the viral components necessary for the formation and function of viral particles. However, in most cases, multiple plasmid expression vectors or individual expression cassettes stably integrated into host cells are used to isolate the various genetic components that generate viral vector particles.

[0102] In some embodiments, expression cassettes encoding one or more viral packaging and envelope proteins were stably incorporated into mammalian cells. In these embodiments, transduction of the viral vectors described herein into these cells is sufficient to produce viral particles without the addition of further expression vectors.

[0103] In other embodiments, the in vitro method includes the use of multiple expression vectors. In some embodiments, the method includes transduction into mammalian cells one or more expression vectors encoding viral packaging proteins and envelope proteins necessary for particle formation.

[0104] The ssDNA AAV genome contains two open reading frames, Rep and Cap, adjacent to two 145-base terminal inversion sequences (ITRs) that form the basis for the synthesis of complementary DNA strands. Rep and Cap produce several proteins (Rep78, Rep68, Rep52, Rep40, and the capsid proteins VP1, VP2, and VP3, which are necessary for the AAV lifecycle). Transgenes are inserted trans-intercepted between the ITRs and Rep and Cap. The AAV2 backbone is commonly used and described in Srivastava et al., J. Virol., 45:555-564 (1983). Cis-acting sequences that direct viral DNA replication (ori), packaging (pkg), and integration into the host cell chromosome (int) are contained within the ITRs. AAV also requires helper plasmids containing adenovirus-derived genes. These genes (E4, E2a, and VA) mediate AAV replication. Examples of pAAV plasmids are available from Addgene (Cambridge, MA, USA) as plasmid numbers 112865 or 60958.

[0105] After the release of the virus particles, the culture medium containing the virus particles may be recovered, and the virus particles may be separated from the culture medium if necessary. The virus particles may also be concentrated if necessary.

[0106] After production and any concentration, the viral particles may be stored, for example, by freezing at -80°C, in preparation for use by administration to cells and / or use in therapy.

[0107] The present invention also provides viral particles produced, for example, by the methods described herein. When used herein, viral particles include a DNA or RNA genome packaged within a viral envelope that can infect cells, such as mammalian cells. Viral particles may be integrase-deficient and may include, for example, a mutant integrase enzyme or modifications to the 5' and / or 3'LTR as described herein.

[0108] In another embodiment, the present invention provides a capsid encoded by a nucleotide sequence encoding a capsid as described herein. In some embodiments, the capsid comprises an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NOs. 12, 14, 15, or 16.

[0109] In another embodiment, the present invention provides a viral particle comprising a capsid encoded by a nucleotide sequence encoding a capsid as described herein. In some embodiments, the capsid comprises an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NOs. 12, 14, 15, or 16.

[0110] Methods for restoring or treating neuromuscular or neuromotor disorders AAV capsids identified by the screening methods described herein may be used to develop gene therapies for treating various conditions or disorders.

[0111] Accordingly, one aspect of the present invention provides a method for restoring or treating a neuromuscular state or neuromuscular disorder or a neuromotor state or neuromotor disorder in a subject, the method comprising administering a therapeutically active amount of the AAV expression vector or viral particles of the present invention to the subject.

[0112] Another aspect of the present invention provides a method for restoring or treating neuromuscular or neuromotor disorders in a subject, the method comprising administering a therapeutically active amount of the AAV expression vector or viral particles of the present invention to the subject, wherein the AAV expression vector or viral particles comprises a nucleotide sequence encoding the capsid of an AAV capsid identified by a screening method of the present invention, and the iPSC or ESC used in the screening method is derived from the subject. In some embodiments, the iPSC or ESC used in the screening method is derived from a skin sample of the subject.

[0113] As a result, in some embodiments, the AAV capsid is "individually configured" for the subject, and the treatment method becomes specific to the subject being treated ("personalized"). One way of examining the "individuality" of the AAV capsid identified by the screening method of the present invention is as follows: (i) IPSCs are collected from one mouse strain (e.g., C57 / Bl6), and these cells are screened for AAV capsids. (ii) Once the gene sequences of capsids capable of infecting these C57 / Bl6 motor neurons are identified, these same capsids are tested in vivo for infection of genetically identical mice (other C57 / bl6 mice) or genetically similar mice (e.g., BALB / c and CD1). Each of these strains of mice is injected intramuscularly with the AAV capsid. (iii) Examine the number of infected motor neurons in each lineage. Previous studies have shown that AAV capsids evolved in one lineage do not efficiently infect other lineages (Horeaux et al, 2018). If the AAV capsids evolved in the screening method of the present invention are so, this would indicate that AAV capsids evolved from individual patients are the most efficient for gene therapy.

[0114] In some embodiments, the method includes retrogradely infecting neurons with an AAV expression vector or viral particle for the purpose of delivering genetic material to neurons, for the purpose of treating neuromuscular disorders or neuromotor disorders or disorders affecting movement, or for the purpose of targeting neurons for any therapeutic purpose.

[0115] In some embodiments, the above method includes altering the activity of neurons in the subject.

[0116] In some embodiments, AAV expression vectors or viral particles are delivered intramuscularly to retrogradely infect motor neurons of the subject's neurons and alter the activity of motor neurons within the subject.

[0117] As used herein, “retrograde transport” or “retrograde infection” means the uptake of the vector at the axon terminal (or “synaptic terminal”), i.e., the synapse, and its transport through the axon to the neuron body in the opposite direction to the direction of action potential propagation (hence “retrograde”). Subsequently, the viral nucleic acid enters the nucleus, where it can replicate and become transcriptionally and translationally active.

[0118] Such delivery is beneficial when the nerve cell body and / or the axon itself are inaccessible, but the region of the terminal process, including the synapse, is available for gene vector delivery. Therefore, if a gene vector capable of retrograde transport can be successfully delivered to such a region of the terminal process, retrograde transport and infection of vulnerable projection neurons may occur.

[0119] Once a virus is transported to the neuron body, its nucleic acid is typically localized in the cell nucleus. According to some embodiments of the present invention, adeno-associated virus particles that undergo retrograde transport to the neuron body can directly insert their nucleic acid contents into the nucleus.

[0120] Embodiments of the present invention include the delivery of a substantially non-toxic recombinant adeno-associated virus vector having the heterologous gene of interest to provide retrograde gene delivery to a neuronal cell body resulting in gene expression.

[0121] Many different neurological disorders impair quality of life by affecting movement. The last common pathway from the nervous system to the muscles may be that of motor neurons, and these neurons are affected in many different disorders, only a few of which are classified as “motor neuron disorders.” In some embodiments, the AAV expression vector of the present invention can access motor neurons after intramuscular injection and subsequently treat neuromuscular or neuromotor disorders by modifying gene expression in the motor neurons, leading to healing, symptom relief, and / or improved quality of life for patients with diseases affecting motor neurons.

[0122] As used herein, “neuropathy” means any disorder that causes morphological and / or functional abnormalities in a nerve cell or population of nerve cells. Neuropathy may impair or eliminate normal nerve function in a subject, or cause abnormal nerve function to be present. For example, neuropathy may be a result of disease, injury, and / or aging. Non-limiting examples of morphological and functional abnormalities include physical deterioration and / or death of nerve cells, abnormal growth patterns of nerve cells, abnormal physical connections between nerve cells, underproduction or overproduction of substances by nerve cells, such as neurotransmitters, failure of nerve cells to produce substances they normally produce, production of substances, such as neurotransmitters, and / or generation or transmission of electrical impulses in abnormal patterns or at abnormal times.

[0123] As used herein, “neuromotor disorder” typically refers to a developmental or acquired disorder affecting motor / gross motor ability, posture, and fine motor skills. This disorder is caused by damage to the central nervous system, which may result from problems or damage related to the development of developing motor pathways in the cortex, basal ganglia, thalamus, cerebellum, brainstem, spinal cord, or peripheral nerves. The most common neuromotor disorders in childhood include cerebral palsy, muscular dystrophy, and spina bifida. The most common neuromotor disorders in adults include stroke, multiple sclerosis, Parkinson's disease, and trauma. This functional disorder may be static (not worsening) or progressive.

[0124] In some embodiments, neuromuscular or neuromotor disorders are spasticity. As used herein, “spasticity” refers to a condition in which a particular muscle is persistently or abnormally contracted. This contraction can cause muscle rigidity or tension, interfering with the normal movement of the face, limbs, trunk, and / or sphincter muscles, and potentially resulting in defects in, for example, speech, gait, and / or bladder and bowel function. Spasticity is a condition that occurs in widespread CNS disorders affecting brain and / or spinal cord function, including, for example, trauma to the brain or spinal cord, multiple sclerosis, cerebral palsy, stroke, or other conditions. Spasticity develops when, despite the presence of underlying symptoms, the characteristics of motor neurons change in accordance with those symptoms, generating excessive electrical impulses and resulting in excessive muscle contraction. This damage alters the balance of signals between the nervous system and muscles, increasing muscle excitability. Spasticity is seen in conditions in which the brain and / or spinal cord are damaged or do not develop normally. These include cerebral palsy, multiple sclerosis, spinal cord injury, and acquired brain injury, including stroke.

[0125] Several mouse models of spasticity have been created, and the ability of the expression vector of the present invention to treat spasticity can be verified using these models. In one embodiment, the ability of the expression vector to treat spasticity resulting from spinal cord transection (see, for example, Yoshizaki et al. 2020) can be tested as follows. (i) Skin biopsy material is collected from a mouse that is genetically identical to the mouse used in the in vivo spasticity experiment. (ii) Using these biopsy materials, IPSCs are prepared and motor neurons are prepared for use in the AAV capsid screening method described herein to select the optimal capsid. (iii) Perform spinal cord transection in the thorax of the mouse. (iv) The severity of spasticity is assessed by behavioral observations such as the Modified Ashworth Scale, or by implanting an EMG recording device into the muscle. (v) After transection, the mice are divided into three groups. Group 1 is intramuscularly injected with an AAV containing the DNA sequence of a gene intended to reduce synaptic firing of motor neurons, using a capsid obtained from the screening process. Group 2 is intramuscularly injected with an AAV having the same capsid as Group 1, but its DNA expresses inactive proteins such as GFP. Group 3 is intramuscularly injected with an AAV (AAV6) having a wild-type capsid containing the same DNA sequence as Group 1. (vi) Monitor changes in spasticity symptoms before and after intramuscular AAV injection.

[0126] This experiment will allow us to investigate whether the in vitro screening process yields AAV capsids that are more efficient at infecting motor neurons than currently available AAV capsids (comparing group 1 to group 3), and whether the viral DNA cargo is sufficient to reduce spastic symptoms (comparing group 1 to group 2).

[0127] Another promising model animal is the pig. In this case, skin biopsy material can be obtained from the animals used in the experiment and screened and experimented on in a similar manner to mice.

[0128] In certain embodiments, the present invention also provides the use of expression vectors and viral particles as described herein for the manufacture of a drug for treating neuromuscular or neuromotor disorders in human or animal subjects, expression vectors as described herein for use in the treatment of neuromuscular or neuromotor disorders in human or animal subjects, and a method for treating neuromuscular or neuromotor disorders comprising administering the expression vectors and viral particles as described herein to an individual in need thereof.

[0129] Dosage and Administration The viral particles and expression vectors described herein can be delivered to a subject in various ways (e.g., intramuscular, intravenous, intracranial, or intraspinal). In some preferred embodiments, the particles and expression vectors described herein can be delivered to a subject via intramuscular injection.

[0130] The specific method and site of administration may be at the discretion of the physician, who will use common general knowledge to select administration methods and methods known to those skilled in the art.

[0131] Following administration of viral particles, recipient individuals may exhibit a reduction in the symptoms of the treated disease or disorder. For example, in treated individuals, recipient individuals may show improved neuronal firing, synaptic release of neurotransmitters, neuronal survival, growth, or connectivity.

[0132] As used herein in the context of treatment of a condition, the term “treatment” generally relates to human treatment and therapy in which some desired therapeutic effect, such as inhibition of the progression of the condition, is achieved. This term includes a reduction in the rate of progression, a cessation of the rate of progression, a regression of the condition, a recovery of the condition, and a cure of the condition. Treatment as a preventative measure (i.e., prevention, avoidance) is also included.

[0133] Viral particles can be delivered in therapeutically effective doses.

[0134] As used herein, the term “therapeutic dose” refers to the amount of viral particles that, when administered according to the desired treatment regimen, is effective in producing some desired therapeutic effect that is commensurate with a reasonable benefit-risk ratio.

[0135] Similarly, the term “preventive dose,” as used herein, refers to the amount of viral particles that, when administered according to the desired treatment regimen, is effective in producing some desired preventive effect commensurate with a reasonable benefit-risk ratio.

[0136] In the context of this specification, “prevention” should not be understood to describe complete success, i.e., complete protection or complete prevention. Rather, in this context, prevention refers to measures taken with the aim of maintaining health by helping to delay, mitigate or avoid a particular condition before it is detected as a symptomatic condition.

[0137] While viral particles can be used alone (e.g., by administration), it is often preferable to provide them as a composition or formulation, for example, together with a pharmaceutically acceptable carrier or diluent. In some cases, the viral particles are administered co-administered with a second expression vector containing an adenovirus helper gene, which may optionally be pHelper.

[0138] As used herein, the term “pharmaceutically acceptable” refers to compounds, components, materials, compositions, dosage forms, etc., that are suitable for use in contact with the tissues of a subject (e.g., human) in a reasonable benefit-to-risk ratio, without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of sound medical judgment. Each carrier, diluent, excipient, etc., must also be “acceptable” in the sense that it is compatible with the other components of the formulation.

[0139] In some embodiments, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicine) that comprises, consists essentially of, or consists of virus particles and a pharmaceutically acceptable carrier, diluent, or excipient as described herein.

[0140] As described in WO2008096268, in embodiments of gene therapy using delivery of virus particles, the unit dose can be calculated with respect to the dose of virus particles administered. The dose of virus includes a specific number of virus particles or plaque forming units (pfu). For embodiments involving adenovirus, a specific unit dose is 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 or 10 14 pfu. The dose of particles may be somewhat higher (10 - 100 fold) because of the presence of non-infectious particles.

[0141] In some embodiments, the methods or treatments of the invention may be used in combination with other therapies, whether symptomatic or disease modifying.

[0142] The term "treatment" includes combinatorial treatments and combination therapies that combine two or more treatments or therapies, e.g., sequentially or simultaneously.

[0143] For example, it may be beneficial to combine treatment with a compound as described herein with one or more other (e.g., 1, 2, 3, 4) agents or therapies.

[0144] Appropriate examples of co-therapeutics will be known to those skilled in the art based on the disclosures herein. Typically, co-therapeutics may be any known in the art that, given the diagnosis of the individual being treated, are thought to be effective in treating the diseases described herein.

[0145] The above-mentioned active agents (i.e., viral particles and one or more other active agents) may be administered simultaneously or sequentially, and may be administered individually via different routes with different administration schedules. For example, when administered sequentially, these active agents may be administered at close intervals (e.g., 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 hours or longer, or, if necessary, even longer intervals), and the exact administration regimen should be in line with the characteristics of the therapeutic agent.

[0146] kit The present invention also provides AAV vectors as described herein, as well as kits comprising one or more viral packaging expression vectors and envelope expression vectors as also described herein. In some embodiments, the viral packaging expression vector is an integrase-deficient viral packaging expression vector.

[0147] cell The present invention also provides cells containing AAV vectors as described herein. In some embodiments, these cells are mammalian cells, such as human cells.

[0148] None of the subheadings in this specification are included for convenience only and should not be construed as limiting the disclosure. The present invention will now be further described with reference to the following non-limiting drawings and examples. Other embodiments of the present invention will be conceivable to those skilled in the art in light of these. All disclosures of references cited herein are expressly incorporated herein by cross-reference, as they can be used by those skilled in the art to practice the present invention. This application claims priority to German patent application 2010981.5, filed on 16 July 2020. The contents of that application are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0149] [Figure 1] A therapeutic approach to treating spasticity. A modified AAV vector is injected into the muscle, infecting the terminals of the motor neurons controlling that muscle (1). The virus is then transported to the cell bodies of motor neurons in the spinal cord, where its genes are expressed, alleviating symptoms (2). [Figure 2] Schematic diagram of an example of a "personalized approach" to AAV capsid screening. Schematic diagram showing a promising pipeline of gene therapy for treating spasticity. (i) Skin samples are taken from the patient, (ii) iPS cells are generated using the samples, and (iii) motor neurons are generated. (iv) AAV libraries are screened in vitro at the terminals of these motor neurons, and (v) effective retrograde capsid sequences are extracted from the motor neuron cell bodies. (vi) AAVs containing genes that alter motor neuron activity are produced in a GMP facility using these synthetic capsid sequences, and (vii) they are used for intramuscular injection in the patient. [Figure 3] Schematic diagram of microfluidic culture of motor neurons and muscle cells. [Figure 4](A) A general scheme for assaying the AAV capsid library against stem cell-derived neurons. (B) Embryonic stem cell-derived motor neurons in culture (expressing GFP under the control of the Hb9 promoter) used for pilot infection with the AAV library. (C) DNA agarose gel showing the capsid band at 2.2Kb. DNA was collected from motor neurons and amplified by PCR. [Figure 5] (A) An example protocol for in vitro reproduction of the neuromuscular junction from motor neurons and muscles. (B) Fluorescence image of neurites passing through a central microchannel in a microfluidic chamber. (C) Fluorescence image showing neurites extending, branching, and contacting muscle fibers. Panels A-C are excerpts from Mills et al., 2018 Molecular Metabolism 7:12-22. D) An example strategy for screening an AAV capsid library in an in vitro model of stem cell-derived motor neurons and neuromuscular junctions. [Figure 6] The mechanism of microfluidic devices. [Figure 7] Restriction of non-retrograde AAVs to the axonal compartment. [Figure 8] Application of an AAV capsid library to the axonal compartment of a microfluidic chamber, and collection / bioinformatics of the resulting capsid sequences. [Figure 9] AAVSeqA-tdTomato in developing motor neurons in microfluidic devices. [Modes for carrying out the invention]

[0150] Examples Example 1 - Identification of capsid sequences that infect motor neurons A general scheme for assaying AAV capsid libraries in stem cell-derived neurons is shown in Figure 4A.

[0151] Using the method disclosed herein, the AAV library was applied to mouse embryonic stem cell-derived motor neurons expressing GFP under the control of the Hb9 promoter (Figure 4B).

[0152] As shown in Figure 4C, the DNA agarose gel showed the presence of a capsid band at 2.2 kb. This DNA was collected from motor neurons.

[0153] A combination of PCR and Sanger sequencing to identify the capsid sequences that infected those motor neurons as Sequence ID 1 and Sequence ID 2.

[0154] Virtual Example 2 - Identification of Capsid Sequences that Infect Subcompartments of Motor Neurons The method of this disclosure can be extended and adapted to identify AAVs in terms of their ability to infect neuronal subcompartments (e.g., synaptic terminals or axons). For example, motor neurons can be grown in a microfluidic chamber that separates the neuronal axon from the cell body. Furthermore, by adding various cell types to this system, in vivo conditions, such as the neuromuscular junction, can be more closely replicated (an example protocol is shown in Figure 5A). Neuritic processes can pass through a central microchannel within the microfluidic chamber, as described in Mills et al., 2018 Molecular Metabolism 7:12-22 (Figures 5B and 5C).

[0155] A general strategy for screening AAV capsid libraries for stem cell-derived motor neurons in an in vitro model of the neuromuscular junction is shown in Figure 5D.

[0156] The screening method may include the following steps: i) Obtain iPSCs or ESCs (collectively referred to as "stem cells") from animals, human subjects / patients, or cell banks. In culture medium, induce these stem cells to develop into a specific class of neuron or other cell type (e.g., motor neurons, sensory neurons, dopaminergic neurons, muscle tissue). ii) Grow these neurons in a culture system (i.e., a microfluidic chamber) that physically separates the nerve cell body from other cellular compartments (i.e., nerve processes, axons). iii) Further cell types (e.g., sensory neurons and muscle cells) can be added to the culture system to more closely match the in vivo environment. iv) Apply the AAV library to one compartment of the culture system (i.e., a compartment containing axons but not cell bodies). v) Collect genetic material from a separate compartment, e.g., a compartment containing the nerve cell body, for the purpose of identifying the capsid sequence that successfully infected these cells via synaptic terminals.

[0157] The obtained gene sequences can be further used to construct rAAV vectors for capsid proteins that utilize those sequences. These vectors can be used for further directional evolution to increase evolutionary pressure, or for the development of gene therapies.

[0158] Example 3 - Co-culture of motor neurons and muscle cells in a microfluidic device Motor neurons cultured in a microfluidic device extend a large number of axons through microfluidic grooves and make contact with myotubes (muscle cells). These motor neurons can be maintained in the culture medium for more than a week (Figure 6).

[0159] The microdevices were sterilized and cleaned with ethanol and mounted on a glass substrate according to the instructions in the user manual (Xona Microfluidics SND150). Each device was named the motor neuron (MN) culture compartment and the myotube culture compartment. First, both cell culture compartments were coated with diluted Matrigel at 37°C for at least 2 hours.

[0160] First, we collected and seeded C2C12 cells (available from ATCC), an immortalized mouse myoblast cell line. The myoblasts were then divided into 12 × 10⁶ cells. 6 The cells were resuspended at a density of cells / ml and pipetted into a muscle culture compartment (12 ml) to obtain 144,000 myoblasts per device. After 24 hours, myoblast differentiation was induced by adding myoblast differentiation medium (DMEM containing 5% horse serum). The myoblasts were differentiated for 48 hours to form multinucleated fibers. After myoblast differentiation, motor neurons were collected and measured at 15 × 10⁶. 6 The cells were resuspended at a density of cells / ml. Motor neurons were then pipetted into an MN compartment (12 ml), yielding 180,000 MNs per device. The devices were incubated at 37°C for 2 hours to promote cell adhesion, after which the respective media were added to fill the devices. Addition of 20 ng / ml of GDNF and BDNF to the muscle compartment, combined with fluid flow from the muscle compartment to the motor neuron compartment using the 20 μl volume difference between the compartments, resulted in neurite recruitment.

[0161] Figure 6 shows a merged image of fluorescence and bright-field images of a growing Hb9-mESC-derived motor neuron (left) and an axon (right) extending through a microsulcus, branching, and in contact with a differentiated myotubule in a Matrigel-coated compartment.

[0162] Example 4 - Restriction of AAV particles into the axon chamber By applying non-retrograde AAV (SEQ ID NO: 13 / 14) to the muscle compartment, AAV infection occurs only in the muscle compartment, demonstrating that AAV particles do not pass through the microfluidic groove (Figure 7).

[0163] Figure 7 shows bright-field images (Figure 7A) and fluorescence images (Figure 7B) of the same motor neuron-microfluidic culture. AAV6, which encodes the red fluorescent protein tdTomato, was added to the axonal compartment. Non-neuronal cells in the axonal compartment expressed tdTomato, indicating that they were infected with AAV. Cells in the motor neuron compartment did not express tdTomato, demonstrating that AAV itself does not pass through the microfluidic barrier.

[0164] Example 5 - Collection of capsid sequences from motor neurons and muscle cells after application of the AAV capsid library. After applying the AAV capsid library to the axonal side of a microfluidic chamber, mutated capsid sequences can be collected from both motor neurons (via retrograde transport) and myotubes (via direct infection) via PCR.

[0165] The AAV6 library was applied to the muscle chamber of a microfluidic device (Figure 8A). Seven days after application, neuronal cell bodies were collected by trypsin treatment. Neurons were lysed and used as PCR templates with primers targeting the conserved region of the AAV6 capsid. After PCR of the capsid region (Figure 8B), the DNA fragment was cloned into a skeletal vector, and the entire process was repeated three times to increase evolutionary pressure. The final PCR product of the capsid region was cloned into a DNA vector and subjected to Sanger sequencing. The results were analyzed using bioinformatics for highly enriched capsids. Muscle cells were also lysed in the final step and used as PCR templates with the same specific primers to find motor neuron enriched capsids that also infect muscle cells (Figures 8C and 8D). Bioinformatics analysis revealed several differences in enriched capsids when comparing infection of motor neurons with infection of muscle.

[0166] Figure 8 shows the application of the AAV capsid library to the axonal compartment of a microfluidic chamber, and the collection / bioinformatics of the resulting capsid sequences. (Figure 8A) Experimental design. (Figure 8B) Representative PCR of AAV capsids against DNA collected from motor neurons or muscle cells. The capsid is shown as a band of approximately 2Kb. (Figure 8C) Bioinformatics analysis of sequences collected from motor neurons. Each column is a distinct sequence, and the black line in the column indicates the difference from the parent AAV6 capsid sequence. (Figure 8D) Similar to (Figure 8C), except for DNA collected from muscle cells.

[0167] Example 6 - In vitro testing of motor neuron-enriched capsid sequences: Co-culture of motor neurons and muscle cells in a microfluidic device After bioinformatics analysis, we selected the more frequently occurring capsid (8% of the sequence), which we named sequence A (sequence number 11 / 12).

[0168] The capsid of sequence A was re-cloned into an ITR2-REP2 vector and packaged into an AAV expressing the td-Tomato fluorescent marker. AAVSeqA-tdTomato was applied to the muscle chamber of a microfluidic device. After 7 days, live imaging (Figure 9) and fluorescence imaging (Figure 9) showed retrograde infection of motor neurons by the novel sequence A capsid AAV virus expressing the tdTomato fluorescent marker.

[0169] Figure 9 shows AAVSeqA-tdTomato in motor neurons growing in a microfluidic device. Fluorescence images of Hb9-mESC-derived motor neurons retrogradely infected with the AAVSeqA-tdTomato virus. Figure 9A shows a merged image with all fluorescence channels merged. Figure 9B shows the tdTomato signaling, Figure 9C shows an H9-GFP motor neuron, and Figure 9D shows the DAPI-stained nucleus.

[0170] References Bedbrook CN, Deverman BE, and Gradinaru V. Viral Strategies for Targeting the Central and Peripheral Nervous Systems. Annual Review of Neuroscience 2018 41:1, 323-348 Buning H, Huber A, Zhang L, Meumann N, Hacker U Engineering the AAV capsid to optimize vector- host-interactions. Current Opinion in Pharmacology, 24, (2015), 94-104, https: / / doi.org / 10.1016 / j.coph.2015.08.002. Dalkara et al., In Vivo-Directed Evolution of a New Adeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery from the Vitreous. Science Translational Medicine 2013:Vol. 5, Issue 189, pp. 189ra76 DOI: 10.1126 / scitranslmed.3005708 Davidsson et al., A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism. Proceedings of the National Academy of Sciences Dec 2019, 116 (52) 27053-27062; DOI: 10.1073 / pnas.1910061116 Deverman et al., Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nature Biotechnology. 2016 34(2):204-9. doi: 10.1038 / nbt.3440. Hordeaux et al., The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL / 6J Mice. Molecular Therapy. 26(3):664-668. doi: 10.1016 / j.ymthe.2018.01.018. Li, C., Samulski, R.J. Engineering adeno-associated virus vectors for gene therapy. Nature Reviews Genetics 21, 255-272 (2020). https: / / doi.org / 10.1038 / s41576-019-0205-4 Pulicherla et al., Engineering liver-detargeted AAV9 vectors for cardiac and musculoskeletal gene transfer. Molecular Therapy. 2011 19(6):1070-8. doi: 10.1038 / mt.2011.22. Tervo et al., A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons. Neuron. 92(2):372-382. doi: 10.1016 / j.neuron.2016.09.021. Vandenberghe, L., Wilson, J. & Gao, G. Tailoring the AAV vector capsid for gene therapy. Gene Ther 16, 311-319 (2009). https: / / doi.org / 10.1038 / gt.2008.170 Yang et al., A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection. Proceedings of the National Academy of Sciences Mar 2009, 106 (10) 3946-3951; DOI: 10.1073 / pnas.0813207106

[0171] Sequence Listing Nucleotide sequence of capsid sequence collected from motor neurons derived from ES / iPSC (SEQ ID NO: 1)

[0172] [Table 1] TIFF0007864110000002.tif24161

[0173] Nucleotide sequences of capsid sequences collected from ES / iPSC-derived motor neurons (SEQ ID NO: 2)

[0174] [Table 2]

[0175] Nucleotide sequence of the KCC2 gene (SEQ ID NO: 3)

[0176] [Table 3] TIFF0007864110000005.tif239163TIFF0007864110000006.tif129162

[0177] Amino acid sequence of the KCC2 gene product (SEQ ID NO: 4)

[0178] [Table 4]

[0179] Kv1 gene nucleotide sequence (SEQ ID NO: 5)

[0180] [Table 5] TIFF0007864110000009.tif239162TIFF0007864110000010.tif239161TIFF0007864110000011.tif43161

[0181] Amino acid sequence of the Kv1 gene product (SEQ ID NO: 6)

[0182] [Table 6]

[0183] Nucleotide sequence of the tetanus toxin light chain gene (SEQ ID NO: 7)

[0184] [Table 7]

[0185] Amino acid sequence of the tetanus toxin light chain gene product (SEQ ID NO: 8)

[0186] [Table 8] TIFF0007864110000015.tif9161

[0187] Nucleotide sequence of the hM4Di gene (SEQ ID NO: 9)

[0188] [Table 9]

[0189] Amino acid sequence of the hM4Di gene product (SEQ ID NO: 10)

[0190] [Table 10]

[0191] Nucleotide sequence of sequence A (SEQ ID NO: 11)

[0192] [Table 11] TIFF0007864110000019.tif72161

[0193] Amino acid sequence of sequence A (SEQ ID NO: 12)

[0194] [Table 12]

[0195] AAV6 capsid DNA sequence (SEQ ID NO: 13)

[0196] [Table 13] TIFF0007864110000022.tif53162

[0197] AAV6 amino acid sequence (SEQ ID NO: 14)

[0198] [Table 14]

[0199] The amino acid sequence of the capsid encoded by SEQ ID NO: 1 (SEQ ID NO: 15)

[0200] [Table 15]

[0201] The amino acid sequence of the capsid encoded by SEQ ID NO: 2 (SEQ ID NO: 16)

[0202] [Table 16]

Claims

1. An in vitro method for screening nucleotide sequences encoding the capsid of adeno-associated virus ("AAV") particles capable of infecting neurons in a test subject, (i) A step of providing a population including neurons, The aforementioned neurons are derived from induced pluripotent stem cells ("iPSCs") or embryonic stem cells ("ESCs"), Each of the neurons has a first specific subcompartment and a second specific subcompartment, in the process, (ii) The step of arranging the neurons such that the first specific subcompartment and the second specific subcompartment are far apart from each other. (iii) The step of bringing the first specific subcompartment into contact with a first plurality of test AAV particles, (iv) the step of isolating a first plurality of AAV particles infected in the second specific subcompartment, and (v) The step of determining the nucleotide sequences encoding the capsids of the first plurality of AAV particles that have infected the second specific subcompartment of the neuron. In vitro methods, including those mentioned above.

2. (a) In step (ii), the first specific subcompartment and the second specific subcompartment are grouped in different physical regions of one or more containers, and / or (b) The first specific subcompartment and the second specific subcompartment are grouped in different physical regions of one or more containers within the microfluidic chamber, and / or (c) The first specific subcompartment and the second specific subcompartment are connected by an axon, and / or (d) The one or more containers further include skeletal muscle cells and / or muscle cells and / or sensory neurons, The in vitro method according to claim 1.

3. The in vitro method according to claim 1 or 2, wherein the neuron is a motor neuron.

4. After the aforementioned decision-making process, (vi) A step of preparing a second set of test AAV particles using the nucleotide sequences encoding the capsids of the first set of AAV particles that have infected the neurons. (vii) Repeating steps (i) and (ii) and bringing the first specific subcompartment into contact with the second plurality of test AAV particles in order to isolate a second plurality of AAV particles that have infected the neurons, and (viiii) A step of determining the nucleotide sequence encoding the capsid of the second plurality of AAV particles that have infected the neuron. It further includes, The nucleotide sequence encoding the capsid of the second plurality of AAV particles is more effective in infecting the neurons than the nucleotide sequence encoding the capsid of the first plurality of AAV particles. The in vitro method according to any one of claims 1 to 3.

5. The second set of test AAV particles described above, Random mutagenesis of the nucleotide sequences encoding the capsids of the first plurality of AAV particles that have infected the neurons, Shuffling of the nucleotide sequences encoding the capsids of the first plurality of AAV particles that have infected the neurons, and Insertion of a targeted peptide sequence or random peptide sequence up to 25 amino acids in length in various regions of VP1, VP2, or VP3 of the nucleotide sequence encoding the capsid of the first plurality of AAV particles that have infected the neurons. The in vitro method according to claim 4, which is produced by one or more of the following.

Images